Staphylococcus aureus is a primary human pathogen and a leading cause of hospital-acquired infections (over 700,000 annually), food poisoning, sepsis, and toxic shock syndrome. Currently more than 90 percent of community-isolated strains of S. aureus are resistant to penicillin or its derivatives. The ubiquity of S. aureus and its ability to rapidly develop antibiotic resistance have prompted monitoring by the WHO, the CDC and others. Understanding the basis for the pathogenicity of S. aureus opens the door to the development of new therapeutics to combat infectious diseases produced by this organism. S. aureus produces a number of virulence factors. Sequencing of strains of S. aureus has shed light into how the genes for these factors are organized. Recent studies have revealed that the genes for a number of the pyrogenic toxin superantigens are located on mobile genetic elements called pathogenicity islands that are about 16 kb in size and flanked by direct repeats. Recent microarray analysis of S. aureus pathogenicity island 3 (SaPI3) from strain MN NJ has shown that mRNA is produced for 21 of the 23 ORFs examined. In SaPI3 only 6 of these open reading frames (ORFs) encode for proteins whose sequences are homologous to proteins with a known structural fold. The goal of this project is to use the structural genomics paradigm to investigate the SaPI3 ORFs. If soluble protein cannot be isolated or crystallized for a particular ORF, orthologs from other pathogenicity islands (6 S. aureus pathogenicity islands have been identified to date) will be expressed and studied. Functional hypotheses derived from the structures and analyses of ORF null mutants will be tested using assays by the principal investigator and his collaborators. The principal investigator has been working on gram-positive pathogens since 1993. Since then workers in the laboratory have determined the structures of staphylococcal toxic shock syndrome toxin-1 (wild type and 8 mutants), of streptococcal pyrogenic exotoxin A, and of staphylococcal exfoliative toxins A and B. Progress toward the goals to this proposal include cloning 22 ORFs of SaPI3, the production of soluble protein from 7 ORFs and the crystallization of proteins from 3 ORFs.